In experimental animal sciences and related industries, techniques for cryopreserving embryos are critical and fundamental for the production of animals or the preservation of cell lines via reproduction technology or the like. In the case of mice, the preservation of 2-cell-stage embryos has been already implemented because of the ease of sampling (see Nakao K. et al., Exp. Anim. 46(3), 231-234, 1997).
When early embryos, such as rat 2-cell-stage embryos, are preserved, however, the rate of retrieval is poor. Such preservation does not yield a rate of fetal development similar to that obtained from untreated embryos (see D. G. Whittingham, J. Reprod. Fert, 1975, 43, 575-578; and M. S. Han et al., Theriogenology 59, 2003, 1851-1863). Thus, there are many issues to be resolved regarding techniques for cryopreserving experimental animal embryos.
In contrast, it has been expected that primate ES cells are put to clinical use, such as the study of development, the preparation of genetically engineered animals, or the regenerative medical techniques. In order to effectively utilize such ES cells for the aforementioned purposes, it is necessary to multiply these cells to a great extent, preserve the multiplied cells, and provide the amount required at the time of use. Further, cell culture usually involves the risk of a mutation of traits, which necessitates preservation of some established cells. Several techniques for preserving primate ES cells have been already reported. A method that yields a high rate of cell survival (see Reubinoff B. E. et al., 2001, Human Reprod. Vol. 16, No. 10, 2187-2194), however, can preserve only approximately 20 to 30 ES cell clumps. A method that can simultaneously preserve a large number of ES cells is available (see Fujioka, T. et al., 2004, Int. J. Dev. Biol., 48: 1149-1154), although the rate of cell survival after thaw is as low as 10% to 20%. Accordingly, development of a method that enables simultaneous preservation of a large number of ES cells, yields a high rate of cell survival after thaw, and allows maintenance of an undifferentiated state has been awaited.
It has been known that direct introduction of a solution with a high content of cryoprotective agent into liquid nitrogen or liquid nitrogen gas results in vitrification of a solution rather than crystallization thereof. The term “vitrification” substantially refers to a phenomenon where a solution becomes solidified instead of being crystallized. A variety of techniques have been developed to preserve animal cells via vitrification. However, there has been no preservation technique for rat early embryos or mammalian ES cells that can yield a high rate of cell retrieval.